U.S. patent application number 17/037162 was filed with the patent office on 2022-06-09 for method and system for detecting analyte of interest using magnetic field sensor and magnetic particles.
The applicant listed for this patent is IMRA America, Inc.. Invention is credited to Matthew L. Elani, Alison R. Garrett, Bing Liu.
Application Number | 20220178919 17/037162 |
Document ID | / |
Family ID | 1000005287078 |
Filed Date | 2022-06-09 |
United States Patent
Application |
20220178919 |
Kind Code |
A1 |
Liu; Bing ; et al. |
June 9, 2022 |
METHOD AND SYSTEM FOR DETECTING ANALYTE OF INTEREST USING MAGNETIC
FIELD SENSOR AND MAGNETIC PARTICLES
Abstract
A method, system, and apparatus for the rapid detection of
analyte(s) of interest are disclosed which can provide high
sensitivity quantification of the analyte concentration in a
lateral follow assay. The method includes labeling detection
molecules with magnetic particles and immobilizing the magnetic
particles on a nitrocellulose membrane upon specific biochemical
recognition and binding. An external magnetic field is applied to
the magnetic particles to induce magnetic induction, and a
magnetoresistance sensor is positioned close to the membrane and
magnetic particles. A periodic signal in the sensor is produced
when a mechanical oscillatory movement is provided to the membrane
relative to the sensor (or vice versa). Triggered time averaging of
signals in synchronization with the oscillatory motion enables
noise reduction of less than 30 dB and significant improvement of
assay sensitivity. An x-y motion program for scanning the test line
and control line on the membrane can produce magnetic 2D mapping of
the lines, further differentiating the bound particles at the lines
from unbound particles in the background, rendering a more accurate
assay.
Inventors: |
Liu; Bing; (Ann Arbor,
MI) ; Elani; Matthew L.; (Ann Arbor, MI) ;
Garrett; Alison R.; (Ann Arbor, MI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
IMRA America, Inc. |
Ann Arbor |
MI |
US |
|
|
Family ID: |
1000005287078 |
Appl. No.: |
17/037162 |
Filed: |
September 29, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62916995 |
Oct 18, 2019 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/76 20130101;
G01N 33/54333 20130101; G01N 33/6887 20130101; G01N 27/745
20130101 |
International
Class: |
G01N 33/543 20060101
G01N033/543; G01N 33/76 20060101 G01N033/76; G01N 33/68 20060101
G01N033/68; G01N 27/74 20060101 G01N027/74 |
Claims
1. A system comprising: an apparatus comprising: at least one
permanent magnet; and at least one magnetic field sensor at a pole
of the at least one permanent magnet and configured to be
positioned sufficiently close to a surface of a membrane containing
immobilized magnetic particles selectively bound to the analyte
such that the magnetic particles are magnetized by the at least one
permanent magnet; a stage mechanically coupled to at least one of
the apparatus and the membrane, the stage configured to move at
least one of the apparatus and the membrane relative to one another
with an oscillatory movement along a first direction substantially
parallel to the surface of the membrane; at least one controller
configured to control the oscillatory movement of the stage and to
generate first synchronization trigger signals indicative of the
oscillatory movement; and a data acquisition unit configured to
receive sensor signals from the at least one magnetic field sensor
and the first synchronization trigger signals from the at least one
controller.
2. The system of claim 1, wherein the stage comprising a linear
motion sub-stage configured to move at least one of the apparatus
and the membrane relative to one another with a linear mechanical
movement in a second direction substantially parallel to the
surface of the membrane, the second direction substantially
perpendicular to the first direction, the at least one controller
further configured to control the movement of the sub-stage along
the second direction and to generate second synchronization trigger
signals indicative of the linear mechanical movement, the data
acquisition unit further configured to receive the second
synchronization trigger signals from the at least one
controller.
3. The system of claim 1, wherein the stage comprises at least one
piezoelectric actuator and/or at least one voice coil actuator.
4. The system of claim 1, wherein the at least one magnetic field
sensor comprises at least one magnetoresistance (MR) sensor.
5. The system of claim 4, wherein the at least one MR sensor
comprises a Wheatstone bridge comprising four individual MR
sensors.
6. A method for qualitative or quantitative measurement of analyte
selectively bound to magnetic particles immobilized on a membrane,
the method comprising: providing an assembly comprising at least
one permanent magnet and at least one magnetoresistance (MR) sensor
attached to one pole of the at least one permanent magnet;
positioning the membrane sufficiently close to the at least one MR
sensor such that the magnetic particles are magnetized by an
external magnetic field generated by the at least one permanent
magnet and the magnetic inductance of the magnetic particles are
detected by the at least one MR sensor; moving, in a periodic
oscillation, the membrane relative to the assembly or the assembly
relative to the membrane, the periodic oscillation in a first
direction substantially parallel to a surface of the membrane;
generating a first trigger signal in synchronization with the
periodic oscillation; acquiring a sensor signal from the at least
one MR sensor and averaging the sensor signal over time in
synchronization with the periodic oscillation, the acquisition and
time averaging being triggered by the trigger signal; and obtaining
a concentration of the analyte.
7. The method of claim 6, further comprising moving the membrane
along a second direction perpendicular to the first direction and
parallel to the membrane surface to form a two-dimensional scanning
motion in a plane parallel to the membrane surface, and generating
a second trigger signal in synchronization with the two-dimensional
scanning motion, wherein said sensor signal is acquired in
synchronization with the two-dimensional scanning motion, and
provides a two-dimensional signal distribution in the plane
parallel to the membrane surface.
8. The method of claim 6, wherein the periodic oscillation has a
frequency in a range of 1 Hz to 100 Hz and an amplitude in a range
of 0.1 mm to 10 mm.
9. The method of claim 8, wherein the periodic oscillation movement
is generated by a piezoelectric actuator and/or a voice coil
actuator.
10. The method of claim 6, further comprising receiving a signal
from a position sensor positioned relative to the membrane or to a
container holding the membrane, the position sensor configured to
monitor a contact or a position between the membrane and the at
least one MR sensor.
11. The method of claim 10, further comprising, in response to the
signal from the position sensor, applying a third motion to the
membrane and/or to the at least one MR sensor in a third direction
perpendicular to the membrane surface to adjust a distance between
the membrane and the at least one MR sensor.
12. The method of claim 6, wherein the magnetic particles are
superparamagnetic, ferromagnetic, or have another form of magnetism
such that the magnetic particles are configured to generate a
magnetic induction upon magnetization by a magnetic field.
13. The method of claim 6, wherein the membrane contains a
two-dimensional array of dots comprising deposited binding
molecules, each dot having a different capture molecule reacting to
a different analyte.
14. The method of claim 6, wherein the membrane comprises paper,
glass, metal, and/or semiconductor.
15. The method of claim 6, wherein positioning the membrane
comprises using a linear motion stage and/or a rotational motion
stage.
16. A system for qualitative or quantitative measurement of analyte
of interest, wherein magnetic particles selectively bind to analyte
and are immobilized on a membrane surface, said membrane is
transported to within a close distance to a magnetoresistance
sensor, whereby said magnetic particles are magnetized by an
external magnetic field generated by a permanent magnet, whereby
the magnetic induction of said magnetic particles are measured by
said magnetoresistance sensor, and the concentration of analyte is
obtained, said magnetoresistance sensor is attached to one pole of
said permanent magnet, forming an assembly, said membrane moves in
a periodic oscillation relative to said assembly of
magnetoresistance sensor and permanent magnetic, or alternatively
and equally effective, said assembly of magnetoresistance sensor
and permanent magnet moves in a periodic oscillation relative to
said membrane, wherein an electric trigger signal is generated in
synchronization with said periodic oscillation movement, said
magnetoresistance sensor signal is acquired and averaged over time
in synchronization with said periodic oscillation, said time
acquisition and averaging being triggered by said electric trigger
signal.
17. The system according to claim 16, wherein said membrane is
transported along a second direction perpendicular to said periodic
oscillation in the plane of membrane, forming a two dimensional
scanning motion in the plane of membrane, where an electric trigger
signal is generated in synchronization with said two dimensional
scanning motion, wherein said magnetoresistance signal is acquired
in synchronization of said two dimensional scanning motion, and
provide two dimensional distribution of signal in the plane of
membrane.
18. The system according to claim 16, further comprising a pressure
sensor below said membrane or a container holding the membrane, the
pressure sensor configured to monitor a contact between the
membrane and the magnetoresistance sensor.
19. The system according to claim 20, wherein a third motion is
provided to said membrane or to said magnetoresistance sensor, the
third motion in a direction perpendicular to the plane of the
membrane to adjust a distance between said membrane and said
magnetoresistance sensor, said third motion is controlled by
feedback signal from said pressure sensor.
20. The system according to claim 16, further comprises a linear
motion stage and/or a rotational motion stage configured to
position said membrane.
21. The system according to claim 16, further comprising an optical
camera positioned above the membrane and configured to record
images and measure color intensity of said magnetic particles,
whereby the concentration of analyte is obtained.
22. An apparatus for qualitative or quantitative measurement of an
analyte of interest, the apparatus comprising: at least one
permanent magnet; and at least one magnetic field sensor at a pole
of the at least one permanent magnet and configured to be
positioned above a surface of a lateral flow membrane containing
immobilized magnetic particles.
Description
CLAIM OF PRIORITY
[0001] This application claims the benefit of priority to U.S.
Provisional Appl. No. 62/916,995 filed on Oct. 18, 2019 and
incorporated in its entirety by reference herein.
BACKGROUND
Field
[0002] This application relates generally to magnetic sensing and
reader systems of biological assays using magnetic particles as
probes.
Description of the Related Art
[0003] Biological binding assays, such as immunoassays and DNA
hybridization assays, use either fluorescent molecules or solid
particles as probes (e.g., signal reporter). The detection
molecules (e.g., antibody or detection DNA) are first labeled with
fluorescent molecules or solid particles, in a process also known
as conjugation. Upon specific recognition and binding with antigen
molecules, the probes are immobilized on a solid surface by the
complementary capturing molecules (e.g., capture antibody or DNA).
The assay results are obtained by measuring the fluorescence
intensity of the bound fluorescent molecules or the color intensity
of the bound solid particles.
[0004] The dominant fluorescence assay in diagnostics is the
Enzyme-Linked Immunosorbent Assay (ELISA). The dominant solid
particle-based assay is lateral flow assay (LFA), where gold
nanoparticles are most commonly used due to their intense red
color. ELISA has the advantage of high sensitivity but requires
extensive technical training and controlled laboratories. Lateral
flow assay is rapid, user-friendly, and very low-cost. Lateral flow
assay can be constructed with low-cost materials such as
nitrocellulose membranes for capillary flow of liquid sample,
plastic backing card, fiber glass sample pad and liquid wick pad.
However, lateral flow assays generally lack high sensitivity and
quantitative results.
SUMMARY
[0005] In certain implementations, a method and system are provided
for rapid and precise quantification of the concentration of
molecules of interest. In certain implementations, the method and
system provide a read out of the magnetic induction field intensity
in the reaction zones (e.g., test line and control line) in a
lateral flow assay using magnetic particles as the probe.
[0006] In certain implementations, an assembly comprises at least
one magnetic (e.g., magnetoresistance) field sensor and at least
one permanent magnet, the at least one magnetic field sensor
attached to a pole of the at least one permanent magnet. The
assembly is configured to be positioned such that the at least one
magnetic field sensor is above a surface of a lateral flow membrane
containing immobilized magnetic particles (e.g., such that the
surface of the membrane is in close proximity to a surface of the
at least one magnetic field sensor). In certain implementations, at
least one of the assembly and the membrane is configured to be
moved relative to the other with a periodic oscillatory mechanical
movement (e.g., the membrane is moved relative to the assembly
while the assembly is stationary; the assembly is moved relative to
the membrane while the membrane remains stationary). The periodic
oscillatory mechanical movement is in a direction that is
substantially perpendicular to a test line and control line of the
membrane, and has an amplitude that is at least twice a width of
the test line and control line. In certain implementations, the
periodic oscillatory mechanical movement has a frequency that does
not cause significant mechanical hysteresis (e.g., time lag) of the
oscillatory motion (e.g., does not cause motion instability that
affects timing precision).
[0007] In certain implementations, the movement is provided by a
mechanical motion stage (e.g., a stage comprising at least one
piezoelectric actuator and/or at least one voice coil actuator). In
certain implementations, a periodic trigger signal (e.g.,
electronic signal) is generated in synchronization with the
periodic oscillatory mechanical movement and the trigger signal is
supplied to a data acquisition (DAQ) unit to synchronize
acquisition and time averaging of the magnetic field sensor signal
with the movement.
[0008] In certain implementations, at least one of the assembly and
the membrane are configured to be moved relative to the other with
at least one second linear mechanical movement (e.g., the
mechanical motion stage is configured to move the membrane for
scanning the assembly in a direction along the test line and
control line). For example, the at least one second linear
mechanical movement can be in two substantially perpendicular
directions (e.g., x-y motion) configured to provide a
two-dimensional (2D) magnetic mapping of the test line and control
line. The intensity of the sensor signal obtained during the 2D
mapping can be summed along the test line and control line to
provide a one-dimensional (1D) profile of the magnetic particle
distribution in a direction substantially perpendicular to the test
line and control line.
[0009] In certain implementations, the at least one magnetic field
sensor comprises a Wheatstone bridge comprising four
magnetoresistance sensors. In certain implementations, the
Wheatstone bridge has a size that is smaller than a width of the
test and control line (e.g., a size sufficiently small to provide
2D spatial resolution in lateral flow assay magnetic mapping).
[0010] In certain implementations, the membrane is held by a
cassette and a position sensor (e.g., mechanical pressure sensor;
proximity sensor) is positioned relative to (e.g., on, near,
inside, below, or beneath) the membrane and/or the cassette, the
position sensor indicative of a contact or a distance between the
membrane and the at least one magnetic field sensor. A sensor
signal generated by the position sensor (e.g., a signal indicative
of a pressure applied by the cassette to the mechanical pressure
sensor) is configured to be supplied as a feedback signal to a
third linear actuator providing vertical movement (e.g., in a
direction that is substantially perpendicular to a plane of the
membrane), the third linear actuator configured to use the feedback
signal to keep the distance between the membrane and the at least
one magnetic field sensor substantially constant.
[0011] In certain implementations, a combined system and a lateral
flow assay are configured to determine a presence and/or absence of
one or more target analytes in a sample (e.g., to
semi-quantitatively or to quantitatively determine an amount of at
least one target analyte in a sample). Examples of target analytes
compatible with certain implementations described herein include
but are not limited to: biomarkers (e.g., antibodies to an
infectious disease, cancer biomarkers, other indicators including
proteins, peptides, nucleic acids, and polysaccharides); infectious
disease agents (e.g., viruses, bacteria, molds); drugs of abuse.
Samples compatible with certain implementations described herein
can be biologically derived (e.g., from humans, animals, plants,
fungi, yeast, or bacteria), or may be derived from food, water,
soil, air, or other sources (e.g., to test for contamination). In
certain implementations, the magnetic probe in this system
advantageously enables analyte detection at concentrations that are
at least one order of magnitude lower than concentrations that can
be detected using a lateral flow assay based on optical sensing.
Such increased sensitivity can be highly valuable in a number of
fields, including but not limited to: point-of-care testing; food
safety; animal health; other fields in which it is advantageous to
quickly identify the presence and/or amount of a target analyte
with a high degree of accuracy. Certain implementations described
herein are configured to be readily integrated with existing
lateral flow assays by exchanging the optical probe particles
(e.g., gold nanoparticles) with magnetic particles (e.g., iron
oxide nanoparticles).
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1A shows an example magnetization curve of magnetic
particles and FIGS. 1B and 1C schematically illustrate
magnetization of magnetic particles using an AC magnetic coil and a
permanent magnet, respectively, with an example positioning of a
magnetoresistance (MR) sensor with respect to the magnetic
particles and the magnetization source.
[0013] FIG. 2A schematically illustrates an example apparatus and
an example system comprising the apparatus in accordance with
certain implementations described herein.
[0014] FIG. 2B schematically illustrates the example apparatus and
another example system comprising the apparatus in accordance with
certain implementations described herein.
[0015] FIGS. 3A-3D show various views depicting an example
apparatus and example system in accordance with certain
implementations described herein.
[0016] FIG. 4 schematically illustrates an example electronic block
diagram of the at least one magnetic field sensor and example
signal processing circuitry (e.g., for signal conditioning and
amplification) in accordance with certain implementations described
herein.
[0017] FIG. 5 shows an example plot of the dependence of the
measured field sensor signal on the sensor height relative to the
membrane surface in accordance with certain implementations
described herein.
[0018] FIGS. 6A-6C are plots of theoretical calculations of the
magnetic AC susceptibility of iron oxide nanoparticles as a
function of frequency for particles of different sizes, assuming
Gaussian distributions with .sigma.=2 nm.
[0019] FIG. 7A schematically illustrates an example trapezoidal
speed profile of a closed loop oscillation system with some
hysteresis (e.g., time lag) at zero speed where the motion reverses
direction in accordance with certain implementations described
herein.
[0020] FIG. 7B schematically illustrates an example train of
magnetic field sensor signals in accordance with certain
implementations described herein in which the test line passes the
sensor at the peak velocity of the oscillation.
[0021] FIG. 7C is a plot of a measured series of mechanical
hysteresis (e.g., time lag) of a hundred oscillations driven by a
piezoelectric oscillation system at 4 Hz in accordance with certain
implementations described herein.
[0022] FIGS. 8A-8D demonstrate the effectiveness of noise reduction
by the y-direction oscillation and triggered averaging in
accordance with certain implementations described herein.
[0023] FIGS. 9A-9C show a demonstration of the high sensitivity of
an assay of human chorionic gonadotropin (hCG) in accordance with
certain implementations described herein and FIG. 9D is a plot of
the FFT spectrum of the voltage signal.
[0024] FIG. 10 schematically illustrates an example geometric setup
and block diagram of the motion program in accordance with certain
implementations described herein.
[0025] FIGS. 11A-11B show two example magnetic 2D mappings of test
lines of an hCG assay with hCG concentrations of 1 ng/ml and 0.1
ng/ml, respectively, in accordance with certain implementations
described herein. FIGS. 11C-11D show two example magnetic 2D
mappings of test lines of a cTnI assay with cTnI concentrations of
1 ng/ml and 0.1 ng/ml, respectively.
[0026] FIG. 12 schematically illustrates a lateral flow assay in
accordance with certain implementations described herein.
[0027] FIG. 13 illustrates a photograph of seven resulting lateral
flow strips of different hCG concentrations.
[0028] FIGS. 14A-14B illustrate plots of the dose response curves
produced using the colorimetric reader and in accordance with
certain implementations described herein, respectively.
[0029] FIG. 15 illustrates a photograph of eight lateral flow
strips of different cTnI concentrations.
[0030] FIGS. 16A-16B show dose response curves produced using the
standard colorimetric reader and the system in accordance with
certain implementations described herein, respectively.
[0031] FIG. 17 illustrates a photograph of three representative
strip at different biotin-BSA concentrations.
[0032] FIGS. 18A-18B illustrate the average magnetic and optical
signals at each concentration, as well as the approximate average
noise signal using the standard colorimetric reader and the system
in accordance with certain implementations described herein,
respectively.
[0033] FIGS. 19A-19B schematically illustrates an example voice
coil linear actuator and an example system utilizing the voice coil
linear actuator, respectively, in accordance with certain
implementations described herein.
[0034] FIGS. 20A-20B show example plots of the time-averaged
background noise on the MR sensor used with a voice coil linear
actuator, magnetic shielding, and axial positioning in accordance
with certain implementations described herein.
[0035] FIGS. 21A-21H show an example series of measurements of hCG
assay taken using a voice coil linear actuator in accordance with
certain implementations described herein.
[0036] FIGS. 22A-22D show another example series of measurements of
Streptavidin/Biotin assay membranes using the voice coil linear
actuator in accordance with certain implementations described
herein.
[0037] FIG. 23 schematically illustrates an example apparatus
having the magnetic field sensor positioned beneath the membrane in
accordance with certain implementations described herein.
[0038] FIGS. 24A-24D show example measurements using a magnetic
field sensor positioned beneath a series of membranes in accordance
with certain implementations described herein.
[0039] The figures depict various implementations of the present
disclosure for purposes of illustration and are not intended to be
limiting. Wherever practicable, similar or like reference numbers
or reference labels may be used in the figures and may indicate
similar or like functionality.
DETAILED DESCRIPTION
Overview
[0040] Detection based on magnetic field sensing and magnetic
particles as probes for lateral flow and other form of assays
(e.g., in situ assays where a sensor is submerged in a sample) can
provide several advantages compared with colorimetric detection of
gold nanoparticles. For example, magnetic sensing is free of
optical interference and can be applied to a wide variety of sample
forms such as whole blood, solid samples, and unprocessed water
samples.
[0041] Magnetic sensing also has the potential of improving
sensitivity in lateral flow assay (e.g., on the order of picograms
to nanograms per milliliter). In traditional colorimetric detection
in lateral flow assay, the signal is based on color intensity
generated by gold nanoparticles. In the reaction zone (e.g., test
line and control line), where the gold nanoparticles are captured
upon specific recognition and binding, only those particles in the
top layer of the nitrocellulose membrane contribute to the color
signal, as the light coming from the particles residing below the
membrane surface are heavily scattered by the porous structure of
the membrane. With magnetic detection, the signal generated by the
magnetic particles residing in the whole depth of the reaction
zones can be detected without scattering loss.
[0042] Lacking quantification (e.g., capability to differentiate at
least between logarithmic steps; e.g., between 1, 0.1, 0.01 ng/ml,
etc.) in lateral flow assay has been an obstacle to full
realization of the potential of this technology. Magnetic sensing
can help improve assay quantification, e.g., by providing
sensitivity at depth below the surface of the test line, and by
scanning and integrating along the test line, such that the entire
population of the magnetic particles captured at the test line can
be measured.
[0043] Applying magnetic sensing to biological assays presents
various challenges. For example, the signals come from small
magnetic particles (e.g., in lateral flow assays, the particle size
is limited by the pores of nitrocellulose membrane to below 200
nm). Because the strength of magnetic induction of a point source
follows an inverse quadratic law with distance, the signal drops
quickly with increasing distance of the magnetic field sensor from
the source, so the magnetic field sensor is to be positioned very
close to the membrane where accessible space is limited.
[0044] For another example, small magnetic particles are
paramagnetic, so the particles do not produce magnetic induction
without an external magnetic field to magnetize them. FIGS. 1A-1C
schematically illustrate the general principle of magnetization of
small particles and methods of detecting them. FIG. 1A shows an
example magnetization curve of magnetic particles, in which the
magnitude of magnetization M of a particle changes with the
external magnetic field H following the hysteresis loop. For very
small particles (e.g., less than 20 nm for iron oxide particles),
the magnetic coercivity H.sub.c diminishes, and the particles
become non-magnetic under zero external field. Such particles are
also known as superparamagnetic and are ideal as probes for
magnetic sensing in biological assays since the particles'
magnetization M and magnetic induction B respond monotonously to
the external magnetization field H, and the particles do not
aggregate in liquid by magnetic dipole interaction.
[0045] To apply an external magnetic field of sufficient strength,
a magnet (e.g., a magnetic coil as schematically illustrated in
FIG. 1B or a permanent magnet as schematically illustrates in FIG.
1C) can be used. To generate strong AC magnetic field, the magnetic
coil uses a high current power supply, and a magnetic core is often
included. A narrow slit can be cut in the core where the magnetic
field is most intense and uniform. Such components are often bulky,
and can conflict with the limited free space available in lateral
flow assay.
[0046] An additional, and potentially more severe, problem is that
although the magnetic induction field of magnetic particles
increases with the external magnetization field strength, the
relatively weak magnetic induction field and the relatively strong
external magnetization field are difficult to distinguish from one
another using the magnetic field sensor (e.g., magnetoresistance or
MR sensor or pickup coils). For DC magnetization, both fields have
similar directions, except for small stray fields from the
particles. For AC magnetization, both fields have the same
frequency and phase.
[0047] A further complication in magnetic sensing for lateral flow
assay is the background signals from unbound magnetic particles.
Although porous nitrocellulose membranes allow fast capillary flow
of the sample liquid, solid particles can be trapped in the pores,
especially when the particles aggregate in liquid. The trapped
particles can become a smeared magnetic background, affecting the
assay sensitivity and accuracy at low antigen concentration.
[0048] Several methods and systems can be used to overcome the
challenges in magnetic sensing for biological assay, depending on
the assay format. For example, in configurations in which the
magnetic field sensor (e.g., magnetoresistance-based; fabricated
into dense arrays for multiplexing detection) can be submerged in a
liquid sample (e.g., in situ assay), the biological reaction can be
realized directly on the sensor surface to minimize the distance
(e.g., less than or equal to 1 micron) between the magnetic
particles and the sensor (see, e.g., U.S. Pat. Nos. 5,981,297 and
9,863,939). The microscopic distance between the magnetic particle
and the sensor surface lowers the strength of the magnetization
field for detection to a value (e.g., to below 50 Oe) that can be
produced by a small coil. However, such a configuration only allows
one-time use of the sensor since the biological molecules are
permanently bonded to the sensor surface and cannot be reused after
the biochemical reaction.
[0049] For lateral flow assays in which the space around the
reaction zones is limited and a macroscopic distance between the
sensor and magnetic particles is unavoidable, a strong external
field is used to magnetize the magnetic particle. For example, a
C-shaped magnet core with a narrow slit can be used to generate an
intense and uniform magnetic field therein (see, e.g., U.S. Pat.
Nos. 6,437,563 and 6,607,922) and a coil wrapped on the back of the
magnetic core can provide high frequency AC modulation. The lateral
flow membrane or a disk containing immobilized magnetic particles
can be inserted into the slit at a close distance to the sensor.
The sensor can include a pair of balanced pickup coils to
differentiate the particles' magnetic induction field from the
strong external magnetization field. To fit into the narrow slit of
the C-shaped magnet, the lateral flow assay cassette containing the
membrane can be a bow-shaped device, where the membrane is
installed as the "string" of the bow without support (see, e.g.,
European Pat. No. EP 1552306).
[0050] Some configurations can utilize multiple high frequency AC
magnetic coils for magnetization and picking up, where pairs of
pickup coils (e.g., each pair including a measurement coil and a
reference coil) are used for differentiating the particles'
magnetic induction field from the external magnetization field
(see, e.g., U.S. Pat. Nos. 6,995,021 and 8,026,716).
[0051] While coil-based magnetization and sensing methods can
provide the advantages of high frequency modulation (e.g., high
signal-to-noise ratio when combined with a lock-in amplifier), a
drawback of certain such configurations is a lack of distinction
between the specific bound magnetic particles in the reaction zone
(e.g., particularly the test line) and nonspecific unbound magnetic
particles trapped in the membrane. Unless the unbound particles are
removed (e.g., by repeated washing), the background signal from the
unbound particles at low antigen concentration can be comparable in
strength to the signal from the specifically bound particles. In
certain implementations described herein, the background signal
from the unbound particles is advantageously reduced by using
measurements with improved spatial resolution so as to spatially
distinguish the reaction zone (e.g., test line) from the
surrounding areas. Such spatial resolution is not provided by
previous coil-based configurations in which the pickup magnetic
coils either cover or enclose the space that includes the samples
(e.g., the test line and a significantly large periphery area).
Previous work using resonant coils in magnetic detection has not
improved spatial resolution (see, e.g., Barnett et al., "An
Inexpensive, Fast and Sensitive Quantitative Lateral Flow
Magneto-Immunoassay for Total Prostate Specific Antigen,"
Biosensors, Vol. 4, pp. 204-220 (2014)).
[0052] In certain implementations described herein, the system
comprises at least one magnetic field sensor based on
magnetoresistance (MR) (e.g., gigantic magnetoresistance (GMR);
tunneling magnetoresistance (TMR)) and the at least one magnetic
field sensor is configured to be applied as a compact magnetic
field sensor for ex situ biological assays where the sensor is
separated in space from the assay (as compared with in situ assays
where the sensor is within the assay liquids, see, e.g., U.S. Pat.
Nos. 5,981,297 and 9,863,939). Progress in semiconductor
manufacturing in the past decades has made such sensors available
for broad applications, beyond their traditional usage as computer
hard disk readers and in automotive speedometers.
[0053] For example, in a previous study of TMR sensors in lateral
flow arrays, a C-shaped permanent magnet with a narrow slit
provided the external magnetization field applied to the magnetic
particles. Similar to the principle of using a balanced pair of
pickup coils in coil-based methods, a pair of TMR sensors were
positioned on both sides of the test line to differentiate against
common mode background signal (e.g., from the external
magnetization field). High frequency AC modulation of the TMR
sensor power supply and a lock-in amplifier were used to improve
the signal-to-noise ratio. An assay of human chorionic gonadotropin
(hCG) demonstrated a limit of detection (LOD) of 25 mIU/ml of hCG
(equivalent to about 2 ng/ml), which is not as good as other
non-magnetic methods, such as a fluorescence-based lateral flow
assay reader (see, e.g., U.S. Pat. No. 9,488,585). Although the
small size of TMR sensors enabled high spatial resolution in
principle, using a sensor pair for common mode rejection rendered
the differential signal as a convolution of two signals from two
sensors separated in space, and sacrificed the special resolution.
(See, e.g., Lei et al., "Contactless Measurement of Magnetic
Nanoparticles on Lateral Flow Strips Using Tunneling
magnetoresistance (TMR) Sensors in Differential Configuration:
Sensors, Vol. 16, p. 2130 (2016)).
[0054] For another example, reader platforms based on GMR sensors
for quantitative lateral flow immunoassays have previously used a
pair of strong permanent magnets (e.g., 4000 G) to provide a
uniform and intense magnetization field, a Helmholtz coil to
provide a sweeping magnetic field at an angle to the pair of
permanent magnets, and a linear (DC) motor system to provide
membrane cassette transportation and distance control between the
sensor and the membrane. Signals were recorded when the membrane
moved relative to the sensor, and stray fields of the magnetic
particles parallel to the membrane plane were measured. Because
such fields are strongest at a distance away from the particles,
the resultant signal had a spatial span of 7 mm in the membrane
plane. This wide span of signal of a single line severely limited
the spatial resolution of detection and caused interference between
the control line and the test line, both of which are used in
standard lateral flow assays and are typically separated by only
5-10 mm (see, e.g., J. Park, "A Giant Magnetoresistive Reader
Platform for Quantitative Lateral Flow Immunoassays," Sensors and
Actuators A, Vol. 250, pp. 55-59 (2016); J. Park,
"Superparamagnetic Nanoparticle Quantification Using a Giant
Magnetoresistive Sensor and Permanent Magnets," J. Mag. And Mag.
Mat'ls, Vol 389, pp. 56-60 (2015)).
[0055] Certain implementations described herein advantageously
utilize magnetic detection for lateral flow and other biological
assays to provide high sensitivity, sufficient specificity against
the background unbound particles, and more reliable quantification
(e.g., capability to differentiate between logarithmic steps; e.g.,
between 1, 0.1, 0.01 ng/ml, etc.) than previous configurations.
Example Implementations
[0056] FIG. 2A schematically illustrates an example apparatus 10
and an example system 100 comprising the apparatus 10 in accordance
with certain implementations described herein. FIG. 2B
schematically illustrates the example apparatus 10 and another
example system 100 comprising the apparatus 10 in accordance with
certain implementations described herein. The apparatus 10
comprises at least one magnetic field sensor 20 (e.g., a
magnetoresistance sensor) and at least one permanent magnet 30, the
at least one magnetic field sensor 20 at (e.g., attached to) a pole
of the at least one permanent magnet 30. The apparatus 10 is
configured to be positioned such that the at least one magnetic
field sensor 20 is above a surface of a lateral flow membrane 40
containing immobilized magnetic particles (e.g., captured in
reaction zones of a test line 42a and control line 42b of the
membrane 40). The apparatus 10 can have an area of contact with the
membrane 40 in a range of 1 mm.sup.2 to 100 mm.sup.2 (e.g., 1
mm.times.1 mm to 10 mm.times.10 mm), and/or is compatible with the
dimensions of standard lateral flow assays (e.g., with membrane
lengths of several centimeters, membrane width of 5 mm, test line
and control line separation of 5 mm to 10 mm). As schematically
illustrated by FIG. 2A, the surface of the membrane 40 is in close
proximity to a surface of the at least one magnetic field sensor 20
(e.g., a distance between the surface of the membrane 40 and the
surface of the magnetic field sensor 20 is in a range of less than
or equal to 100 microns.
[0057] In certain implementations, the at least one magnetic field
sensor 20 comprises at least one magnetoresistance (MR) sensor,
examples of which include but are not limited to: gigantic
magnetoresistance (GMR) sensors and tunneling magnetoresistance
(TMR) sensors. The at least one MR sensor can have a wide range of
dimensions and operation conditions in accordance with certain
implementations described herein. For example, for highly sensitive
applications, bare MR sensors without packaging can be as small as
a few microns. With packaging protection for robust usage, the
dimensions of the at least one MR sensor, including packaging, can
be in a range of 5 mm to 10 mm, and/or is compatible with the
dimensions of standard lateral flow assays. In certain
implementations, the at least one MR sensor can be powered by an
electric voltage in a range of 5 V to 10 V, which consumes a small
amount of electric power, and is therefore compatible with compact
design and portable usage.
[0058] In certain implementations, the at least one permanent
magnet 30 comprises at least one ferromagnetic material, examples
of which include but are not limited to: magnetic materials doped
with rare earth elements, e.g., neodymium; samarium-cobalt. Magnets
compatible with various implementations described herein are widely
available with different shapes, sizes, and strengths. For example,
the permanent magnet 30 can have a size in a range of 5 mm to 10 mm
in each dimension, and/or can have a magnetic field strength in a
range of 500 Oe to 1000 Oe.
[0059] In certain implementations, the lateral flow membrane 40 is
a representation of a plural of solid supporting materials for
immobilization of magnetic particles upon specific detection of
analyte. Other solid materials that can be used to support the
biological recognition reactions include but are not limited to:
paper, glass, metal, and semiconductor. For example, a glass
surface can be printed with antibodies or DNA molecules that bind
specifically to a specific analyte. Gold surfaces are well known to
have good affinity with proteins and antibodies. Assays established
on such solid surfaces with magnetic particles can be detected with
the method and system in accordance with certain implementations
described herein. For another example, the membrane 40 can comprise
nitrocellulose paper with printed capture antibody lines for
capturing antigens (e.g., cut to a rectangular shape of 5
cm.times.50 cm). Liquid capillary flow capacity can be measured by
the time for water to travel 4 cm along the membrane 40, and such
time can be in a range of 80 seconds to 150 seconds. In certain
implementations, the magnetic particles comprise (i) one or more
magnetic materials configured to be magnetized and to generate a
magnetic induction field in response to an applied external
magnetic field and (ii) one or more surface coating materials that
are configured to selectively bind to an analyte of interest to be
detected (e.g., measured) in an assay of the analyte. For example,
the magnetic particles can comprise magnetic iron oxide particles,
which are widely used in biological separations and are compatible
with certain implementations described herein. In certain
implementations, the magnetic iron oxide particles have a size in a
range of 20 nm to 200 nm and/or a magnetic susceptibility in a
range of 20 emu/g to 100 emu/g, and have a surface coating (e.g.,
antibodies for biological recognition and binding). For another
example, the magnetic particles comprise nanoparticles can include
Au--Fe alloy nanoparticles and/or can be fabricated using various
processes (see, e.g., U.S. Pat. Appl. Publ. No. 2011/0192450; Int'l
Publ. No. WO2014/160844; Int'l Publ. No. WO2018/022776; each of
which is incorporated in its entirety by reference herein). Such
Au--Fe alloy particles have a magnetic susceptibility (e.g., in a
range of 50 emu/g to 150 emu/g) that is higher than the magnetic
susceptibility of iron oxide particles, so Au--Fe particles also
have higher magnetic moments and generate stronger magnetic
induction than do iron oxide particles. In certain implementations,
in addition to being configured to generate a magnetic induction
field, the magnetic nanoparticles are further configured to absorb
light (e.g., in the visible range) and to generate colorimetric
signals (e.g., color intensity) to be detected (e.g., measured) in
an assay for the analyte of interest. For example, most iron oxide
particles have a broad optical absorption in the visible range and
have a dark brown color. Magnetic Au--Fe alloy particles have a
broad and nearly flat optical absorption in the entire visible
range and appear nearly black against the white background of the
membrane 40.
[0060] In certain implementations, as schematically illustrated by
FIGS. 2A-2B, the example system 100 further comprises a mechanical
motion stage 50 (e.g., an x-y-z stage) mechanically coupled to at
least one of the apparatus 10 and the lateral flow membrane 40. The
stage 50 is configured to move at least one of the apparatus 10 and
the membrane 40 relative to one another with a periodic oscillatory
movement. For example, the stage 50 can be configured to move the
membrane 40 relative to the apparatus 10 (e.g., while the apparatus
10 is stationary) in a direction that is substantially
perpendicular to the test and control lines 42a, 42b (denoted
herein as the y-direction). For another example, the stage 50 can
be configured to move the apparatus 10 relative to the membrane 40
(e.g., while the membrane 40 is stationary) in a direction that is
that is substantially perpendicular to the test and control lines
42a, 42b (denoted herein as the y-direction). In certain
implementations, the periodic oscillatory mechanical movement has
an amplitude in the y-direction (e.g., in a range of 2 mm to 5 mm)
that is at least twice a width of the test line 42a and/or the
control line 42b (e.g., at least twice a width of 1 mm), but less
than the distance between the two lines 42a, 42b (e.g., to avoid
interference) (e.g., less than a distance in a range of 5 mm to 10
mm). In certain implementations, the periodic oscillatory
mechanical movement has a frequency in a range of 1 Hz to 10 Hz and
a peak linear speed of oscillation (e.g., in the y-direction) in a
range of 10 mm/s to 200 mm/s.
[0061] Various mechanisms (e.g., translation stages) for the
periodic oscillatory mechanical movement are compatible with
certain implementations described herein. Factors to be considered
include but are not limited to: oscillation frequency, amplitude,
range of motion, precision of motion (e.g., minimum hysteresis or
time lag), electric and magnetic noise, device compactness,
lifetime, and cost. Example mechanisms compatible with certain
implementations described herein include but are not limited to:
quartz oscillators, piezoelectric oscillators, piezoelectric
actuators; piezoelectric motors; linear stepper motors, DC motors,
and voice coils. In certain implementations described herein, the
mechanism is selected or designed to advantageously provide
oscillatory movement while reducing (e.g., avoiding; preventing;
minimizing) one or more deleterious effects (e.g., severe
hysteresis; quick wear of gears or other components; high
noise).
[0062] For example, the stage 50 can comprise a voice coil
configured to provide the oscillatory movement. The voice coil can
comprise a magnetic coil and a permanent magnet that can oscillate
freely against each other when an AC current is provided to the
coil. In certain such implementations, the magnetic components of
the system 100 (e.g., the magnetic coil and the opposite magnet of
the voice coil) generate a periodic background signal in the
magnetic field sensor 20 to be filtered out either electronically
or digitally.
[0063] For another example, the stage 50 can comprise a
piezoelectric oscillator configured to provide oscillatory movement
with sufficient frequency (e.g., up to 5 Hz; up to 10 Hz; in a
range of 1 Hz to 100 Hz), sufficient amplitude (e.g., up to 5 mm),
wide range of motion (e.g., up to 20 mm), and low electric and
magnetic noise. In certain such implementations, the piezoelectric
oscillator has a higher hysteresis than does a voice coil, and the
hysteresis can be minimized with a proper set of proportional,
integral, and derivative (PID) parameters in the closed loop
control system.
[0064] In certain implementations, as schematically illustrated by
FIGS. 2A-2B, the stage 50 comprises a linear motion sub-stage 52
(e.g., linear stepper and/or DC motor stage) configured to move or
scan at least one of the apparatus 10 and the membrane 40 relative
to one another with a linear mechanical movement in a direction
substantially parallel to the test and control lines 42a, 42b
(denoted herein as the x-direction). For example, the sub-stage 52
can be configured to move or scan the membrane 40 relative to the
apparatus 10 (e.g., while the apparatus 10 is stationary) in a
direction that is substantially parallel to the test and control
lines 42a, 42b, or the sub-stage 52 can be configured to move or
scan the apparatus 10 relative to the membrane 40 (e.g., while the
membrane 40 is stationary) in a direction that is substantially
parallel to the test and control lines 42a, 42b. In certain
implementations, the movement in the x-direction can be slower than
the movement in the y-direction (e.g., the speed of the x-direction
movement can be in a range of 1 mm/s to 10 mm/s. In certain
implementations, the sub-stage 52 is configured to transport the
membrane 40 to and from the measurement position (e.g., beneath the
apparatus 10). In certain implementations, the stage 50 comprises
the linear motion sub-stage 52 configured for scanning and a
rotational sub-stage configured for transporting the membrane 40 to
and from the measurement position.
[0065] In certain implementations, as schematically illustrated by
FIG. 2A, the example system 100 further comprises a first
controller 60 (e.g., a microprocessor circuit comprising a first
function generator) configured to control the oscillatory movement
of the stage 50 along the y-direction and a second controller 62
(e.g., a microprocessor circuit comprising a second function
generator) configured to control the movement of the sub-stage 52
along the x-direction. In certain implementations, the first
controller 60 is separate from the second controller 62, while in
certain other implementations, the first controller 60 and the
second controller 70 are components of the same circuitry.
[0066] In certain implementations, as schematically illustrated by
FIG. 2A, the example system 100 further comprises a data
acquisition (DAQ) unit 70 (e.g., comprising a microprocessor
circuit). Both the first controller 60 and the second controller 62
can be configured to generate and transmit synchronization trigger
signals to the DAQ unit 70, and the DAQ unit 70 can be configured
to receive the synchronization trigger signals and sensor signals
generated and transmitted by the at least one magnetic field sensor
20 (e.g., via a signal processing circuitry 72 as schematically
illustrated by FIG. 2A). In certain implementations, the triggered
time averaging of the sensor signals is in synchronization with the
movement of the stage 50 in the x- and y-directions, and is
processed by a computer (not shown) running a corresponding
program.
[0067] The example system 100 schematically illustrated by FIGS.
2A-2B can be modified in accordance with certain implementations
described herein. For example, instead of being moved with a
periodic oscillatory movement in the y-direction, at least one of
the apparatus 10 and the membrane 40 can be moved relative to one
another with an periodic oscillatory movement in the x-direction
and instead of being moved with a linear movement in the
x-direction, at least one of the apparatus 10 and the membrane 40
can be moved relative to one another with a substantially linear
movement in the y-direction.
[0068] In certain implementations, as schematically illustrated by
FIG. 2B, the system 100 is configured to perform colorimetric
reading in addition to the magnetic reading of the test and control
lines 42a, 42b. For example, the system 100 can comprise a camera
74 (e.g., an optical camera) configured to be positioned above the
membrane 40 (e.g., before the membrane 40 is positioned below the
at least one magnetic field sensor 20 for magnetic reading. The
images (e.g., photographs) obtained by the camera 74 can provide
color intensity information regarding the test and control lines
42a, 42b. For example, when the analyte concentration is high such
that the test line color is visible and readily detectable by the
colorimetric reading using the camera 74, the color intensity
information can be sufficient for the assay. When the analyte
concentration is low such that the test line color is not visible
and/or readily detectable by the colorimetric reading using the
camera 74 (e.g., the assay can benefit from more sensitive
detection), the system 100 can utilize the apparatus 10 to provide
more sensitive and quantitative assay results from the membrane 40
in accordance with certain implementations described herein.
[0069] FIGS. 3A-3D show various views depicting an example
apparatus 10 and example system 100 in accordance with certain
implementations described herein. FIG. 3A shows a cassette 44
containing the lateral flow membrane 40 in accordance with certain
implementations described herein. In certain implementations, two
sides of the cassette 44 are cut down to the level of the membrane
40 so that the cassette 44 can be transported smoothly to and from
the measurement position below the apparatus 10 (e.g., below the
magnetic field sensor 20). In certain implementations, the cassette
44 can be modified in other ways, or omitted, to allow for such
smooth transportation. As illustrated by FIG. 3A, a cassette holder
46 can be configured to hold the cassette 44 and to be mounted on
the stage 50. In certain implementations, a position sensor (e.g.,
pressure sensor; proximity sensor) (not shown) may be mounted
(e.g., hidden) inside the cassette holder 46 beneath the cassette
44 and configured to monitor the position (e.g., distance) of the
membrane 40 relative to the magnetic field sensor 20 (e.g., by
monitoring the pressure between the membrane 40 and the magnetic
field sensor 20). In certain such implementations, a signal from
the position sensor can be used to maintain sufficient proximity
between the membrane 40 and the magnetic field sensor 20 (e.g.,
close and constant contact without tearing the fragile membrane
40).
[0070] FIG. 3B schematically illustrates an example apparatus 10 in
accordance with certain implementations described herein. The
example apparatus 10 comprises the magnetic field sensor 20 and the
permanent magnet 30 and a metal shielding box 80 containing a
printed circuit board (PCB) 82 comprising the signal conditioning
and preamplifier circuits of the signal processing circuitry 72.
The shielding box 80 can be installed on a y-z stage 90 (e.g.,
manually-controlled and/or electronically-controlled) for precise
height adjustment (denoted herein as the z-direction) and
positioning of the magnetic field sensor 20 relative to the
membrane 40 in the y-direction. The sensor signal from the magnetic
field sensor 20 is transmitted to the DAQ unit 70 via the signal
processing circuitry 72.
[0071] FIGS. 3C and 3D show a right-side view and a perspective
view, respectively, of the example system 100 in accordance with
certain implementations described herein. In FIG. 3C, the shielding
box 80 is denoted by a dashed line. The linear motion sub-stage 52
can comprise a motor (e.g., stepper motor; DC motor) and can be
configured to scan the membrane 40 in a direction along (e.g.,
substantially parallel to) the test and control lines 42a, 42b
(e.g., along the x-direction) and/or to transport the membrane 40,
cassette 44, and cassette holder 46 in and out of the measurement
location for sample exchange (e.g., along the x-direction). The
stage 50 can comprise a piezoelectric motor configured to oscillate
the membrane 40 in a direction substantially perpendicular to the
test and control lines 42a, 42b (e.g., along the y-direction) and
can be mounted on top of the linear motor of the sub-stage 52.
[0072] FIG. 4 schematically illustrates an example electronic block
diagram of the at least one magnetic field sensor 20 and example
signal processing circuitry 72 (e.g., for signal conditioning and
amplification) in accordance with certain implementations described
herein. The at least one magnetic field sensor 20 of FIG. 4
comprises four individual magnetic field sensors 20a-20d in a
Wheatstone bridge configuration. The at least one magnetic field
sensor 20 is configured for common mode rejection such that when an
oscillating membrane 40 periodically passes over the Wheatstone
bridge, the Wheatstone bridge generates a differential signal
pertinent to the movement of the membrane 40. In certain
implementations, the size (e.g., width in a plane substantially
parallel to the membrane 40) of the Wheatstone bridge is configured
to provide spatial resolution in 2D mapping of the test and control
lines 42a, 42b. For example, the size of the Wheatstone bridge can
be smaller than the linewidth of the test and control lines 42a,
42b (e.g., in a range of 0.1 mm to 0.5 mm). In certain
implementations, the signal processing circuitry 72 comprises a
high pass filter circuit having a first cutoff frequency (e.g., 0.3
Hz) configured to remove the DC baseline and a low pass filter
circuit having a second cutoff frequency (e.g., 300 Hz) configured
to remove high frequency noise. The dashed circle of FIG. 4
encircles a TMR sensor of the at least one magnetic field sensor 20
and the enlarged view at the left-side of FIG. 4 schematically
illustrates the orientation of the TMR sensor with respect to the
external magnetic field from the permanent magnet 30. While FIG. 4
schematically illustrates an example implementation using a TMR
sensor (e.g., having a sufficiently high sensitivity for detection
of the magnetic particles of the membrane 40), other types of
magnetoresistance sensors (e.g., GMR sensors) can also be used in
accordance with certain implementations described herein.
[0073] FIG. 5 shows an example plot of the dependence of the
measured sensor signal on the sensor height relative to the
membrane surface in accordance with certain implementations
described herein. An inverse quadratic drop of the measured sensor
signal with increasing distance is observed, which is expected for
small particles as the source of the magnetic field. In certain
implementations, to allow for ample signal detection, the distance
between the sensor surface and the membrane surface is in a range
of less than a few hundred microns (e.g., 0 to 100 microns).
[0074] In certain implementations, the magnetization field from the
permanent magnet 30 is substantially constant in time (e.g., does
not change), and modulation of the signal is performed by
modulating the magnetic induction of the magnetic particles of the
membrane 40, thus improving the signal-to-noise ratio. For example,
a periodic oscillatory motion of the membrane 40 relative to the
apparatus 10, or vice versa, is used (e.g., without using a sensor
pair to differentiate the particles' magnetic signal from the
external magnetization field). For example, the stage 50 (see,
e.g., FIG. 3) can have an oscillation frequency in a range of 1 Hz
to 10 Hz and an amplitude in a range of 1 mm to 5 mm. Other (e.g.,
higher) oscillation frequencies are also compatible with certain
implementations described herein, although the frequency can be
limited by the mechanical capability of the stage 50.
[0075] Compared with the high frequency AC modulation (e.g., kHz)
used previously, low frequency mechanical modulation may initially
appear to be impractical for improving the signal-to-noise ratio,
especially considering the omnipresent 1/f noise. However, a
careful examination of the effect of a linear motion of the
membrane 40 relative to the magnetic field sensor 20 reveals a
better situation, and in certain implementations, low frequency
modulation of the magnetization is both feasible and favored for
magnetic sensing in lateral flow assay. For example, assuming a
sinusoidal motion along the y-direction of y(t)=A sin(2.pi.ft), the
corresponding velocity of the motion is dy/dt=2.pi.Af cos(2.pi.ft).
For a frequency of 4 Hz and an amplitude of 2 mm (e.g., in
accordance with certain implementations described herein), the peak
velocity is about 50 mm/s. Assuming a linewidth of 1 mm for a
lateral flow test line 42a, such an oscillatory motion at its peak
velocity produces a pulsed signal of time width .DELTA.t=0.02 s.
Fourier transformation of the pulsed signal gives a frequency
bandwidth of .DELTA.f=1/.DELTA.t of 50 Hz, which is much better
than the fundamental oscillation frequency f for reduction of 1/f
noise. In certain implementations, the maximum velocity is selected
based on the distance between the test line 42a and the control
line 42b (e.g., in a range of 5 mm to 10 mm) so as to provide
acceleration and deceleration sufficient to distinguish the two
lines 42a, 42b from one another.
[0076] More careful examination of the magnetization behavior of
small magnetic particles in the frequency domain shows that low
frequency magnetic modulation is favored for magnetic sensing in
lateral flow assay. The magnetization curve of small magnetic
particles denoted in FIG. 1A is not representative of AC
magnetization, and the more proper theory of AC magnetization of
small particles is the Neel relaxation theory (see, e.g., B.
Fischer et al., "Brownian Relaxation of Magnetic Colloids," J. Mag.
And Mag. Mat'ls, Vol. 289. Pp. 74-77 (2005)). The Neel relaxation
theory considers the time scale of a small particle's magnetic
moment relaxation under thermal equilibrium, and concludes that the
magnetic moment relaxation time, and therefore the frequency
response in AC magnetization, depends on the particle size.
[0077] FIGS. 6A-6C are plots of theoretical calculations of the
magnetic AC susceptibility (X=M/H) of iron oxide nanoparticles as a
function of frequency for particles of different sizes (e.g.,
diameters) (FIG. 6A: 10 nm; FIG. 6B: 15 nm; FIG. 6C: 20 nm),
assuming Gaussian distributions with .sigma.=2 nm. The real part of
the susceptibility X.sub.r is responsible for the magnetic
induction of particles, while the imaginary part X.sub.i
contributes to heat generation. In FIGS. 6A-6C, both X.sub.r and
X.sub.i are normalized to DC values of 20 nm iron oxide particle.
FIGS. 6A-6C show that except for very small particles of 10 nm, the
susceptibility X.sub.r of magnetic particles drops quickly with
frequency, diminishing at high frequency greater than 1 kHz. These
calculations imply that smaller magnetic particles are advantageous
for AC magnetization. However, since the total magnetic moment of a
particle is proportional to its volume, larger particles (e.g.,
diameters greater than 10 nm) are advantageously used as magnetic
probes for lateral flow as long as these larger particles can move
through the pores of the membrane 40 (e.g., about 200 nm on
average). In view of the drop of susceptibility with frequency for
larger particles (e.g., diameters greater than 10 nm), in certain
implementations, smaller frequencies (e.g., frequencies less than 1
kHz) can be advantageously used for such particles. In certain
implementations, the particle size and frequency are selected
(e.g., optimized) to provide a desired performance (e.g.,
susceptibility; mobility through the membrane pores).
[0078] In certain implementations, the bandwidth of the signal
relies on the peak velocity (e.g., the timing) of the mechanical
oscillation. FIG. 7A schematically illustrates an example
trapezoidal speed profile of a closed loop oscillation system with
some hysteresis (e.g., time lag) at zero speed where the motion
reverses direction in accordance with certain implementations
described herein. In a single oscillation period, the time span of
the peak speed is only a portion of the period, so certain
implementations described herein have the test line 42a pass
through the magnetic field sensor (e.g., apparatus 10) at a peak
speed of the oscillation to maximize the signal. In certain such
implementations, the signal is a regular train of pulses in
synchronization with the speed profile, as schematically
illustrated in FIG. 7B.
[0079] In certain implementations, time averaging of the sensor
signal is triggered by a trigger signal generated by the
oscillation motion controller (e.g., first controller 60) in
synchronization with the oscillation. Such triggered time averaging
is equivalent to lock-in amplification for the purpose of noise
reduction, and in certain implementations, mechanical hysteresis
(e.g., time lag) is reduced (e.g., minimized) by optimizing the
closed loop control parameters (e.g., PID) for precise timing of
averaging, such that peak broadening, which lowers the
signal-to-noise ratio and spatial resolution in the y-profile of
the test and control lines 42a, 42b, is reduced (e.g., prevented).
FIG. 7C is a plot of a measured series of hysteresis of a hundred
oscillations driven by a piezoelectric oscillation system at 4 Hz
in accordance with certain implementations described herein. The
plot of FIG. 7C shows an average mechanical hysteresis of 2-3 ms,
which results in about 1% error in timing, and is acceptable in
certain implementations described herein.
[0080] FIGS. 8A-8D demonstrate the effectiveness of noise reduction
by the y-direction oscillation and triggered averaging in
accordance with certain implementations described herein. For FIGS.
8A-8D, a blank lateral flow strip (e.g., a lateral flow strip
without running any assay and free of magnetic particles) was
tested to examine the background electric noise. FIG. 8A is a plot
of a raw signal, displaying the typical 1/f type of noise
superimposed with high frequency noise. FIG. 8B is a plot of the
fast Fourier transform (FFT) noise spectrum of the raw signal,
where the noise level at 50 Hz is -65 dB. FIG. 8C is a plot of the
FFT noise spectrum of the signal after 1000 averages, where the
noise level at 50 Hz drops to -95 dB, illustrating the
effectiveness of the noise reduction scheme in certain
implementations described herein. FIG. 8D is a plot showing the
quick drop of the root mean square (RMS) value of noise with an
increasing number of averages, which plateaus after 1000 times.
With an oscillation frequency of 4 Hz, the measurement takes 4 min
to finish, which is sufficiently fast for a rapid test.
[0081] FIGS. 9A-9C show a demonstration of the high sensitivity of
an assay of human chorionic gonadotropin (hCG) in accordance with
certain implementations described herein. FIG. 9A is a photograph
of a test strip with an hCG concentration of 0.1 ng/ml in the
sample solution. At the concentration of 0.1 ng/ml, the lateral
flow test line 42b is completely invisible to human eyes. FIG. 9B
is a plot of the raw sensor signal dominated by 1/f noise and high
frequency noise, with the test line signal deeply buried in the
noise. FIG. 9C is a plot of the recovered test line signal,
obtained by following the noise reduction techniques described
herein. To be consistent with optical sensors, such as photodiodes,
whose output is linearly related to energy, the signal reported
herein is the electric power in watts, calculated as V.sup.2/R,
where V is the voltage signal amplitude and R is the sensor's
impedance. The scheme of mechanical oscillation modulation and
synchronized time averaging in certain implementations described
herein works satisfactorily such that the recovered test line
signal is a clean sharp peak as shown in FIG. 9C. FIG. 9D is a plot
of the FFT spectrum of the voltage signal, showing a clear peak in
the frequency domain, peaking at about 25 Hz, with a half width of
about 50 Hz, and a good signal-to-noise ratio of 30 dB. The method
in accordance with certain implementations described herein can
detect down to 0.01 ng/ml of hCG. Such sensitivity is superior by
two orders of magnitudes to the hCG assays previously reported,
which include both TMR sensing (see, e.g., Lei et al. cited herein)
and fluorescence sensing (see, e.g., U.S. Pat. No. 9,488,585).
[0082] In certain implementations, the linear stepper (or DC) motor
sub-stage 52 also provides scanning motion along the test and
control line (e.g., along the x-direction). The advantage of the
small size of MR sensors 20 in obtaining spatially resolved signals
is demonstrated by a programmed x-y scanning of the membrane 40
relative to the magnetic field sensor 20. FIG. 10 schematically
illustrates an example geometric setup and block diagram of the
motion program in accordance with certain implementations described
herein. The membrane 40 has a test line 42a and a control line 42b.
Magnetization field H is perpendicular to the plane of the membrane
40. In certain implementations, the motion control program
automatically searches for the control line 42b in the upper half
of the membrane 40 by determining the location of the signal peak
along the y-direction. The membrane 40 then moves stepwise along
the x-direction while the stage 50 provides periodic oscillation to
produce an x-y scan of the sensor along the control line 42b. The
program then moves the membrane 40 by a defined distance to the
position of the test line 42a and performs the x-y scan along the
test line 42a. The scan speed, the step size, and/or the number of
triggered averages can vary depending on the signal intensity. In
certain implementations, the result is a 2D mapping of the signal
of the test and control lines 42a, 42b, which is unavailable with
previous magnetic sensing systems in lateral flow assay. As
described herein, magnetic particles can be trapped in the lateral
flow membrane pores, contributing to nonspecific background
signals. Certain implementations described herein advantageously
distinguish between bound particles at the test line 42a from the
unbound particles in the background, thereby improving the assay
specificity, reliability, and quantification.
[0083] FIGS. 11A-11B show example magnetic 2D mappings of test
lines 42a of a hCG assay with hCG concentrations of 1 ng/ml and 0.1
ng/ml, respectively, in accordance with certain implementations
described herein. FIGS. 11C-11D show two example magnetic 2D
mappings of test lines of a cTnI assay with cTnI concentrations of
1 ng/ml and 0.1 ng/ml, respectively, in accordance with certain
implementations described herein. The scan in the x-direction
(e.g., along the test line 42a) moves in steps of 0.1 mm, each step
being given 50 triggered averages in synchronization with the
oscillation in the y-direction (e.g., perpendicular to the test
line 42a). The gray scale represents the signal intensity, the
scales of which (in units of electric power) are shown by the scale
bars. The smearing background in FIG. 11B outside the test line
region (marked by the two dotted lines) are likely due to unbound
magnetic particles trapped in the membrane 40. In the cTnI assay,
an additional washing step is adopted to reduce (e.g., remove;
chase away) the unbound magnetic particles after the assay. As a
result, FIG. 11D of the same antigen concentration shows negligible
smearing background. The test line of FIG. 11A was visible to the
naked eye, while the other three test lines of FIG. 11B-11D were
invisible to the naked eye. More details of the two assays,
including photos of the membranes, are described in the sections
describing Example 1 and Example 2. Also, while the number of
averages of 50 in these examples does not fully achieve the noise
reduction capacity demonstrated in FIG. 8D, a larger number of
averages can be used in certain implementations if higher
sensitivity is desired (e.g., with correspondingly longer
acquisition time). These examples highlight that, in certain
implementations, magnetic sensing is highly sensitive to low
antigen concentrations where the test lines are invisible to the
naked eye, and can show features previously unavailable (e.g.,
unbound magnetic particles trapped in the membrane and/or
non-uniformities in test line preparation such as during printing
of the capture antibody), thus helping lateral flow assay
developers identify hidden factors and assay manufacturers improve
quality control.
[0084] The 2D mapping method described herein is not limited to
applications measuring the test and control lines 42a, 42b in
lateral flow assay. In a plural of assay formats, magnetic
nanoparticles can be immobilized on 2D arrays of dots spotted on a
solid surface. The advantage of 2D array of dots are that each dot
can be designed to have biological detection specific to a
different analyte, providing multiplexed assays.
Assay Examples
[0085] The following description provides information regarding
various components of certain implementations described herein,
including the sample, detection and capture ligands, and magnetic
probe, as well as examples of assays and comparisons with standard
colorimetric lateral flow readers.
[0086] FIG. 12 schematically illustrates a lateral flow assay in
accordance with certain implementations described herein. The
components include a membrane 40, detection and capture ligands,
magnetic probes, a wicking pad, a backing card, a sample pad, and a
conjugate pad. FIG. 12 also schematically illustrates a target
analyte, which may or may not be present in a sample of interest.
The lateral flow assay can also be housed within a cassette 44 or
holder to keep all the components together and facilitate flow. In
certain implementations, only the membrane 40, detection and
capture ligands, and magnetic probes are included for the assay,
while certain other implementations also include a wicking pad and
a backing card, and may include the other components as
warranted.
[0087] During testing, a sample (as described herein) is either
applied to the sample pad or directly onto the membrane (e.g. by
dipping the membrane 40 into the sample) and the liquid is pulled
up through the assay via capillary action maintained by the
presence of the wicking pad. If the conjugate pad is present, the
sample can then encounter the magnetic probe functionalized with a
detection ligand. If there is no conjugate pad in the assay, the
magnetic probe can be mixed in with the sample along with a buffer.
If analyte is present in the sample, it can bind to the magnetic
probe to form a binding complex (as described herein). As the
binding complex continues to move through the assay, it can be
immobilized at a capture zone by the capture ligands therein, as
described herein, and the assay can be analyzed before or after the
assay has dried.
[0088] The lateral flow assay can have one or more capture zones.
In some implementations, there can be two capture zones. The first
capture zone can be designed to indicate the presence or absence of
a target analyte (e.g., test line), and the second capture zone can
be designed to indicate that the assay is operational (e.g.,
control line). For example, in a sandwich assay format, as shown in
FIG. 12, the test line capture zone can comprise an immobilized
capture ligand specific to the target analyte. In certain
implementations, there can be two test lines which are each
designed to indicate the presence or absence of a distinct (e.g.,
different) target analyte. Other possible assay formats for
assaying multiple target analytes simultaneously can be used in
other certain implementations.
[0089] The lateral flow assay can include a sample which may or may
not contain an analyte of interest. Herein, the terms "target
analyte," "analyte of interest," and the like refer to a molecule
or moiety that may have some significance when present in the
sample. For example, a target analyte can be a DNA fragment from a
pathogen that may contaminate food. The target analyte can include
many molecules depending on the intended application of the lateral
flow test. For example, for human or animal diagnostic use, the
target analyte can be a biological molecule, such as an antibody or
other protein; a peptide; a nucleic acid, including single- and
double-stranded DNA and RNA, and their fragments (e.g., oligos); a
polysaccharide; a small molecule such as an inhibitor or hormone;
or a combination thereof, such as a protein/RNA complex. In certain
implementations, identifying the presence or absence of such
molecules in a sample can be indicative of diseases, infectious or
otherwise, or other conditions which can impact the body, such as
pregnancy or genetic mutations. As another example, in testing for
contamination of food, water, soil, air, or other environmental
material, the analyte of interest can include foodborne pathogen
markers, such as viral RNA; small molecules, including toxins and
organic compounds; or heavy metals. Certain implementations
advantageously quickly identify environmental hazards. As a final
example, the analyte of interest can be synthetically-derived, as
with testing for drugs of abuse. In each of these examples, the use
of a lateral flow assay can be desirable because it can enable
faster results (e.g., time of measurement less than 6 minutes) than
conventional testing methods.
[0090] The sample applied to the lateral flow strip may or may not
contain the analyte of interest. The sample may or may not be
biologically derived and may contain many distinct (e.g.,
different) molecules or moieties aside from the target analyte. In
certain implementations, the sample can include material derived
from humans, animals, plants, fungi, yeast, bacteria, tissue
culture, viral cultures, or combinations thereof. The sample can
also include extractions from food, water, soil, air, or other
environmental material, or can include extractions from synthetic
materials. Examples of human-derived samples include but are not
limited to: whole blood, serum, plasma, urine, stool, saliva, cheek
swabs and other tissue samples, perspiration, and more. In certain
implementations, the sample can be manipulated in some way to make
it compatible with the lateral flow assay format, or to remove
interfering molecular entities. For example, whole blood can be
filtered so that only serum is applied the membrane, or food
samples can be dissolved so that their component molecular entities
can flow up the strip. The sample can also be modified with
additives, which can either be added directly to the sample before
testing or included on the sample pad. Such additives can be used
to regulate pH (e.g., buffers), to support antibody binding (e.g.,
salts), or to minimize non-specific interactions (e.g.,
surfactants, blockers), among other purposes.
[0091] In certain implementations, the sample can comprise
components artificially mixed to replicate one or more of the
clinically, environmentally, or otherwise relevant samples listed
herein. Certain implementations use such an artificial sample as a
tool for assay development. In such samples, the concentration of
the target analyte can be controlled in order to test possible
assay outcomes. As with other samples, artificially-derived samples
can include many distinct molecules or moieties aside from the
target analyte. In the case of assay development, such inclusions
can provide information about cross-reactivity or interference
caused by their presence.
[0092] The detection and capture ligands can be any molecules,
whether biologically or synthetically derived, which can strongly
and/or specifically bind to the analyte of interest. In certain
implementations described herein, the detection ligands refer to
those analyte binding molecules attached to the probes (e.g., the
magnetic particle probes). The process of attaching detection
ligands to the probes is also known as conjugation or labeling. The
capture ligands refer to those analyte binding molecules localized
(e.g., by printing) at the capture zones on the membrane 40, where
specific binding of analyte occurs complementary to the detection
ligand-analyte binding. As used herein, the terms "specifically
bind," "specific binding," and the like have their reasonable
ordinary meanings, including but not limited to that one binding
molecule or moiety can preferentially bind to a second molecule or
moiety relative to other molecules or moieties in a solution or
sample. For example, an antibody can specifically bind a certain
antigen.
[0093] The exact composition of the detection and capture ligands
can depend on the analyte of interest and the type of assay (e.g.
sandwich, competitive, etc.). In a competitive assay, a detection
ligand can simply comprise the analyte of interest. In various
implementations, the assay can follow the convention of a sandwich
assay where the detection and capture ligands bind to the analyte
of interest complementarily.
[0094] In certain implementations, the detection and capture
ligands can be one member of a binding pair. As used herein, the
term "binding pair" has its reasonable ordinary meaning, including
but not limited to a pair of complementary molecules or moieties
that specifically bind to one another and form a binding complex.
The second member of the binding pair can be the analyte, the
magnetic probe, or other assay components, or can be used to modify
those components. In this way, the binding pair can be used to form
a binding complex between the analyte, the magnetic probe, and the
assay. The binding complex can then allow the analyte to be
detected in accordance with certain implementations described
herein. Examples of suitable binding pairs include but are not
limited to: antibody/antigen pairs, ligand/receptor pairs,
enzyme/substrate pairs, biotin/avidin, biotin/streptavidin, and
antigen- or ligand-binding fragments of antibodies or receptors.
The binding pair for a given assay can be determined by the analyte
of interest and the type of assay.
[0095] In certain implementations, the detection and capture
ligands can be antibodies, and the target analyte can be an
antigen. In a sandwich assay, the capture antibody can bind to one
epitope of the antigen which is complementary to (e.g.,
non-overlapping) the epitope which binds to a detection antibody on
the magnetic probe. In this way, the antigen can bind to both the
magnetic probe and the capture ligand and thus be detected at the
test line.
[0096] The magnetic probes used in certain implementations
described herein are magnetic particles, with magnetic properties
as described herein. The magnetic probes can be conjugated with
detection ligands in order to specifically bind to the target
analyte. In certain implementations, the conjugation can comprise
one member of a binding pair, as described herein as the detection
ligands. The capture ligand printed at the capture zones can be
part of a complementary binding pair to that of the detection
ligand, allowing the target analyte to be bound to both the capture
ligand and detection ligand on the magnetic probe.
[0097] In certain implementations, the magnetic probe can be
functionalized with specific binding moieties (e.g., detection
ligands) via covalent binding. Such process is also referred to as
functionalization. Covalent binding can be achieved either by
reaction of the binding moiety with the magnetic probe surface or
by reaction of the binding moiety with functional groups (e.g.,
--COOH) that have previously been added to the magnetic probe's
surface. Covalent functionalization can be stable over a wide range
of assay conditions. In other implementations, specific binding
moieties can be stably and non-covalently associated with the
magnetic probe surface under the assay conditions. Non-covalent
association mechanisms can include non-specific adsorption,
electrostatic interactions, hydrophobic interactions, hydrogen
bonding interactions, or combinations thereof.
[0098] In some implementations, the surface of the magnetic probe
can be modified prior to functionalization with a specific binding
moiety. In some instances, the surface of the magnetic probe can be
coated or functionalized with a layer designed to facilitate the
functionalization with a specific binding moiety. For example, a
layer of dextran, polyethylene glycol (PEG), or other similar
substance can facilitate the association of a specific binding
moiety with the magnetic probe surface. As another example,
polymers end functionalized with carboxylic groups can be bound to
the magnetic probe surface to allow an EDC/NHS reaction to
covalently bind a protein (e.g., streptavidin). In some
implementations, the surface can be additionally modified with a
surfactant configured to improve the solubility of the magnetic
probe. In some implementations, the surface of the magnetic probe
can be modified with a passivating layer, such as polymers, or
small proteins such as bovine serum albumin (BSA), with the
intention of improving the chemical stability of the magnetic probe
(e.g., prevent aggregation).
[0099] In certain implementations, the magnetic probe can be
blocked after conjugation with a specific binding moiety in order
to reduce or prevent non-specific interactions. As used herein,
"non-specific interactions" has its reasonable ordinary meaning,
including but not limited to binding between assay components
(e.g., magnetic probe and capture ligand) which are not intended to
interact. Non-specific interactions can lead to false test results
(e.g., false positives in a sandwich assay format) and are
therefore avoided inasmuch as possible. Blocking of the magnetic
probe can involve the association of an additional, non-reactive
molecule or moiety with the probe's surface. Example blocking
moieties include but are not limited to: bovine serum albumin
(BSA), Tween-20, Triton X-100, casein, "irrelevant" immunoglobulins
(e.g., immunoglobulins that do not bind to other assay components),
fish skin gelatin, polyethylene glycol (PEG), nonspecific serum
(e.g., horse or fish), commercial blockers, or others, including
combinations thereof. The optimal blocking formulation can be
determined empirically during assay development.
[0100] In some implementations, the magnetic probe can be applied
to a conjugate pad. For example, the magnetic probe can be applied
in a solubilized state and then dried, with the intention that the
magnetic probe will re-solubilize immediately when it comes in
contact with the sample solution. The magnetic probe solution can
be applied to the conjugate pad via spraying, pipetting, dipping,
or other methods. In certain implementations, the magnetic probe
solution that is applied can contain a low concentration buffer for
pH control and a low concentration of a carbohydrate to enhance
re-solvation. The optimal contents, application volume, and
application method may be determined empirically during assay
development.
[0101] Several representative lateral flow assays can be used to
demonstrate the improved sensitivity of certain implementations
described herein over conventional optical-based assay readers
known as colorimetric readers. The term "limit of detection (LOD)"
in these examples has its reasonable ordinary meaning, including
but not limited to the lowest concentration at which a target
analyte can be detected and at which a sample or solution can be
unequivocally distinguished from a solution without the target
analyte. The term "sensitivity" has its reasonable ordinary
meaning, including but not limited to that concentration inversely,
e.g., a higher sensitivity refers to capability of detecting lower
concentration. In some cases, the magnetic probe can bind to the
capture zone when the target analyte is not present, called
non-specific binding in these examples. A large amount of
non-specific binding can lead to decreases in the overall
sensitivity of an assay, because it makes a true positive test more
difficult to distinguish.
Example 1. hCG Assay
[0102] In Example 1, a dose response curve for the pregnancy
indicator human chorionic gonadotropin (hCG) was produced. A dose
response curve is a plot of assay signal versus antigen
concentration which can be used to determine an assay's sensitivity
or to quantify the amount of antigen present in a sample. The
sensitivity can be defined by the concentration below which the
assay signal reaches a saturation point, e.g., the signal does not
change or changes very little at the next lowest concentration.
[0103] A half-strip lateral flow assay was designed and assembled
as follows. The assay included a membrane, two antibody capture
zones, antibody-functionalized magnetic nanoparticles, a backing
card, and a wicking pad. The nitrocellulose membrane had a
capillary flow rate of 120 s (e.g., to flow a distance of 4 cm).
The first capture zone was an anti-hCG primary antibody and the
second was a goat anti-mouse secondary antibody. Each capture zone
antibody solution was applied to the membrane at a concentration of
1 mg/mL. They were sprayed in two lines across the strip with 1 cm
between them. Subsequent preparation steps of baking, blocking,
drying, assembly with backing cards and wick pads, cutting, etc.
followed the standard practice in lateral flow assay.
[0104] Antibody-conjugated magnetic gold-iron alloy (Au--Fe)
nanoparticles were used as the magnetic probe in this assay. The
average size of the magnetic nanoparticles was 150 nm, measured by
dynamic light scattering (DLS). The nanoparticle solution had a
particle mass concentration of 3 mg/ml. Surface functionalized
magnetic nanoparticles were mixed in a 1:1 volumetric ratio with a
1 mg/mL solution of a mouse anti-hCG primary antibody complementary
to that used for the first capture zone. After the reaction, the
nanoparticles were blocked with bovine serum albumin (BSA). The
nanoparticle-antibody conjugates were centrifuged to remove excess
reactants and re-suspended in a buffered solution.
[0105] To produce a dose response curve, seven solutions containing
varying concentrations of the antigen hCG were made, from 100 ng/mL
to 0 ng/mL. The hCG was diluted in a buffer containing a small
amount of Tween-20, designed to reduce or prevent nanoparticle
aggregation and non-specific binding. 50 .mu.L of each antigen
solution was pipetted into a separate well of a 96-well plate. 10
.mu.L of the conjugated magnetic particles (at .about.1.2 mg/mL)
were then added to each well. One lateral flow strip was placed
into each well such that the membrane was partially submerged in
the mixture and the wicking pad stuck up out of the well. The
liquid was allowed to run up the strip for 15 minutes, and then the
strip was removed and allowed to dry for at least 1 hour before
further analysis.
[0106] FIG. 13 illustrates a photograph of the seven resulting
lateral flow strips of different hCG concentrations. The gray line
in the lower half of the strip is the first capture zone (e.g., the
test line), which binds the hCG antigen and is visible only when
antigen of sufficient concentration is present and binds to the
magnetic probe. The dark line in the upper half of the strip is the
second capture zone (e.g., the control line) which binds directly
to the antibody-functionalized magnetic nanoparticles and is used
to indicate that the test is functioning properly. As can be seen,
the visibility of the test line quickly drops off as the hCG
concentration decreases. At 1 ng/ml, the test line is barely
visible to the naked eye. At 0.1 ng/mL, the color can barely be
detected by a colorimetric detector (see below).
[0107] The assay test strips were measured using both a standard
colorimetric lateral flow reader and the magnetic reader in
accordance with certain implementations described herein. FIGS.
14A-14B illustrate plots of the dose response curves produced using
the colorimetric reader and in accordance with certain
implementations described herein, respectively. The curve produced
with the colorimetric reader plateaus at a concentration of 0.01
ng/mL, thus the limit of detection (LOD) is 0.1 ng/mL, or 100
pg/mL. The curve produced in accordance with certain
implementations described herein shows no plateau over the
concentrations examined. The signal at 0 ng/mL is approximately the
same as that at 0.001 ng/mL (0.05 .rho.W). The signal at both of
these concentrations can be attributed to nonspecific binding of
the magnetic particles to the test line. Since the signal at 0.01
ng/mL (0.1 .rho.W) is significantly higher than the nonspecific
binding signal, it is considered the LOD of this hCG assay by the
system in accordance with certain implementations described herein,
illustrating that the sensitivity of this assay has been improved
by one order of magnitude when compared to a standard colorimetric
reader, and two orders of magnitude when compared to the naked
eye.
[0108] The system in accordance with certain implementations
described herein has a broader dynamic range of signal of five
orders of magnitude compared with the two orders of magnitude from
the standard colorimetric reader system. The nonspecific binding
signals of the system in accordance with certain implementations
described herein (e.g., at 0.001 ng/ml and 0 ng/ml) still have a
signal-to-noise ratio greater than 10 (e.g., greater than 20). In
the colorimetric reader system, the test lines of these strips are
completely beyond detection. Therefore, the sensitivity of the
system in accordance with certain implementations described herein
is limited by the imperfect binding chemistry that leaves
nonspecific binding of magnetic particles at the test line, rather
than by the system in accordance with certain implementations
described herein.
Example 2. cTnI Assay
[0109] In Example 2, a system in accordance with certain
implementations described herein was used to produce a dose
response curve for the cardiac injury biomarker cardiac troponin I
(e.g., cTnI). cTnI and another subunit of the cardiac troponin
complex, cTnT, have been established as markers for the diagnosis
of acute myocardial infarction (e.g., heart attack) as well as
other cardiac injuries. Currently, high-sensitivity cardiac
troponin assays have a limit of detection of around 0.01 ng/mL,
allowing for the detection of myocardial injury within 1 to 3 hours
of symptom onset. Less sensitive assays can only be able to detect
such injuries within 3 to 6 hours of symptom onset. Thus, it can be
advantageous to have a limit of detection on the order of 0.01
ng/mL for a cTnI assay to be clinically relevant. However, such
sensitivity is difficult to achieve in a lateral flow assay
utilizing optical measurements. Herein, it is shown that the
sensitivity of a cTnI lateral flow assay can be improved using
magnetic detector particles in accordance with certain
implementations described herein.
[0110] Half-strip lateral flow assay strips were produced as
described above in Example 1 with several changes. The
nitrocellulose membrane had a capillary flow rate of 80 s (e.g.,
for a distance of 4 cm). The first capture zone (e.g., the test
line) was an anti-cTnI primary antibody, applied at a concentration
of 1.5 mg/mL, and the second capture zone (e.g., the control line)
was a goat anti-mouse secondary antibody, applied at a
concentration of 1 mg/mL. The two lines were printed 1 cm apart.
Subsequent preparation steps of baking, blocking, drying, assembly
with backing cards and wick pads, cutting, etc. followed the
standard practice in lateral flow assay.
[0111] Magnetic gold-iron alloy (Au--Fe) nanoparticles were
conjugated with anti-cTnI primary antibodies and were used as the
magnetic probe in this assay. Surface-functionalized magnetic
nanoparticles were incubated in a 1:1 volumetric ratio with a 1
mg/mL solution of a mouse anti-cTnI primary antibody complementary
to that used for the first capture zone. After the reaction, the
particles were blocked with bovine serum albumin (BSA), then
centrifuged to remove excess reactants.
[0112] For the dose response curve, eight solutions each containing
a different concentration of the cTnI antigen were prepared via
serial dilutions, from 100 ng/mL to 0 ng/mL. The cTnI was diluted
in a running buffer containing a small amount of Tween-20, designed
to reduce or prevent non-specific binding. 50 .mu.L of each antigen
solution was pipetted into a separate well of a 96-well plate. 5
.mu.L of the conjugates (at about 1 mg/mL) were then added to each
well and the mixture was stirred with a pipette tip. One lateral
flow strip was placed into each well with the membrane partially
submerged and the wicking pad sticking up out of the well. The
liquid was allowed to run up the membrane for 10 minutes. The
strips were then removed and immediately transferred to a new well
containing 50 .mu.L of the running buffer containing no antigen.
This chase step was included to help reduce non-specific binding of
the magnetic probes to the test line and to the membrane. After 10
more minutes, the strips were removed and allowed to dry for at
least 1 hour before further analysis.
[0113] FIG. 15 illustrates a photograph of the eight lateral flow
strips of different cTnI concentrations. The line in the bottom
half of the strip is the test line, and the line in the top half of
the strip is the control line. The visibility of the test line is a
quick measure of the presence and amount of cTnI antigen present in
the sample. Visually, the test line can only be distinguished with
the naked eye down to a concentration of 10 ng/mL.
[0114] The strips were each analyzed with both a standard
colorimetric lateral flow reader and a system in accordance with
certain implementations described herein. Each strip was measured
once with the colorimetric reader. The strips were measured three
times each with the system in accordance with certain
implementations described herein and the signals were averaged at
each cTnI concentration.
[0115] FIGS. 16A-16B show dose response curves produced using the
standard colorimetric reader and the system in accordance with
certain implementations described herein, respectively. The
colorimetric reader has a limit of detection of 1 ng/mL for this
system (see, FIG. 16A), as the signals for the lower concentrations
are nearly equal to each other and quite close to the lower
detection limit of the device, which varies between 0-15 mV when no
line is visible. The relatively high signal at 0.001 ng/mL may have
been caused by a scratch on the membrane surface, which cannot be
easily distinguished from a test line signal using a colorimetric
reader. One way in which the system in accordance with certain
implementations described herein improves upon such conventional
readers is that the magnetic signal intensity is not affected by
such optical defects.
[0116] The limit of detection of this assay measured using the
system in accordance with certain implementations described herein
is 0.1 ng/mL, as shown in FIG. 16B. In order for a cTnI assay to be
clinically relevant, the limit of detection can be on the order of
0.01 ng/mL. The assay results described herein do not meet this
specification. However, the cTnI assay sensitivity is likely
limited by the chemistry of the assay rather than by the detection
capability of the system in accordance with certain implementations
described herein. In FIG. 16B, the signal to noise ratio in the
plateau region below 0.1 ng/ml is still greater than 20. This
result implies that if the assay chemistry is improved (e.g.,
non-specific binding reduced and/or efficiency of cTnI binding to
the nanoparticles or test line increased), the sensitivity of the
assay can increase. Such improvements would not be available on the
colorimetric reader, as the blank signal (9 mV) is already within
the noise range of that reader (approximately 0-15 mV), and any
optical imperfection such as scratch or stains would disrupt the
color signal. Thus, the system in accordance with certain
implementations described herein may allow for a greater
improvement in sensitivity over the colorimetric reader, given more
thorough development of the assay chemistry.
Example 3. Streptavidin/Biotin Assay
[0117] In Example 3, the system in accordance with certain
implementations described herein was used in conjunction with
half-strip lateral flow assays to evaluate the conjugation
efficiency of streptavidin onto magnetic Au--Fe nanoparticles. The
half-strip assays were produced using the same methods as described
in Example 1, with only the composition and location of the capture
zones differing. In this example, both the control line and test
line included biotinylated bovine serum albumin (biotin-BSA), with
the control line having a significantly higher concentration of
biotin-BSA (0.5 mg/mL). The test line biotin-BSA concentration
varied between 0.1-10 .mu.g/mL. Biotin-BSA was chosen because of
the strong binding interaction between biotin and streptavidin and
the relatively higher molecular weight of BSA, which allows the
molecule to bind more strongly to the nitrocellulose membrane. The
control line and test line were approximately 6 mm apart.
[0118] The magnetic Au--Fe nanoparticles were conjugated with
streptavidin in a similar method as in Examples 1 and 2. The
conjugates were tested using lateral flow by mixing 5 .mu.L of
conjugates with 50 .mu.L of running buffer (1.times.TBS+1%
Tween-20) in one well of a 96-well plate, inserting the half-strip
dipstick assay into it, and allowing capillary flow for 15 minutes.
Three test line concentrations of biotin-BSA were each tested three
times to establish reproducibility: 0.1, 1, and 10 .mu.g/mL. FIG.
17 illustrates a photograph of three representative strip at
different biotin-BSA concentrations. At 1 .mu.g/mL the test line is
barely visible to the naked eye, and at 0.1 .mu.g/mL, the test line
becomes invisible.
[0119] The nine lateral flow strips were measured using both a
conventional colorimetric lateral flow reader and the system in
accordance with certain implementations described herein. Each
strip was measured three times with each device to establish the
variation associated with each reader device. FIGS. 18A-18B
illustrate the average magnetic and optical signals at each
concentration, as well as the approximate average noise signal
using the standard colorimetric reader and the system in accordance
with certain implementations described herein, respectively. The
system in accordance with certain implementations described herein
had a broader dynamic range of signal of five orders of magnitude
above the noise level, as compared to two orders of magnitude for
the colorimetric system. In FIG. 18B, the signal-to-noise ratio of
the magnetic signal at 0.1 .mu.g/ml is greater than 20, while the
signal-to-noise ratio for the colorimetric signal is about 2, which
can be too low to be accepted for a valid measurement. While the
test line of 0.1 .mu.g/ml is barely above the detection limit of
the colorimetric reader, as compared with a blank strip, the
magnetic signal from the same test line is more than an order of
magnitude stronger than the magnetic signal from the blank line,
again proving the superior sensitivity of the system in accordance
with certain implementations described herein.
[0120] FIGS. 19A-19B schematically illustrates an example voice
coil linear actuator 200 and an example system 100 utilizing the
voice coil linear actuator 200, respectively, in accordance with
certain implementations described herein. The voice coil linear
actuator 200 is configured to provide oscillational movement of the
sample membrane 40 relative to the magnetic field sensor 20 of the
apparatus 10 (e.g., in a y-direction substantially perpendicular to
the control and test lines 42a, 42b, as shown in FIG. 19B).
[0121] The example voice coil linear actuator 200 schematically
illustrated by FIG. 19A comprises a permanent magnet 202 extending
within an electrically conductive coil 204 along an axial direction
206 of the coil 204. When an AC electrical current is applied to
the coil 204, an oscillatory motion (e.g., back and forth) along
the axial direction 206 of one or both of the magnet 202 and the
coil 204 is generated in response to the electromotive force (EMF).
In certain implementations, the coil 204 is fixed to a frame (not
shown) and the magnet 202 is free to move in an oscillatory motion,
while in certain other implementations, the magnet 202 is fixed to
a frame (not shown) and the coil 204 is free to move in an
oscillatory motion. In certain implementations, as compared with a
piezo actuator, the voice coil linear actuator 200 advantageously
provides (i) little or no mechanical hysteresis, (ii) higher
operational frequencies (e.g., up to 100 Hz) and speeds (e.g., up
to several hundred mm/s), and/or (iii) longer stroke lengths (e.g.,
up to several centimeters).
[0122] In certain implementations, the voice coil linear actuator
200 has a substantial stray magnetic field which can produce a
strong background signal in the signal generated by the magnetic
field sensor 20. In certain such implementations, the system 100
comprises magnetic shielding configured to reduce the portion of
the stray magnetic field that affects the signal generated by the
magnetic field sensor 20. For example, as schematically illustrated
by FIG. 19B, the voice coil linear actuator 200 can comprise a
magnetic shielding housing 210 at least partially containing the
magnet 202 and the coil 204 and a shaft 220 in mechanical
communication with the stage 50 and with the magnet 202 or the coil
204 that is configured to move. For example, the magnetic shielding
housing 210 can comprise mu-metal (e.g., an alloy made of Ni and Fe
configured to provide magnetic shielding) and the shaft 220 and the
stage 50 can comprise non-magnetic materials (e.g., plastics). In
certain implementations, the voice coil linear actuator 200 can be
positioned to reduce or minimize the effects of the stray magnetic
field on the signal generated by the magnetic field sensor 20. For
example, axial positioning can help further avoid the stray
magnetic fields since the magnetic fields are split into a left
bunch and a right bunch, leaving the center area free of magnetic
field.
[0123] FIGS. 20A-20B show example plots of the time-averaged
background noise on the magnetic field sensor 20 used with a voice
coil linear actuator, magnetic shielding, and axial positioning in
accordance with certain implementations described herein. FIG. 20A
shows the background noise over relatively short time periods
(e.g., less than 0.2 second) and FIG. 20B shows the background
noise over relatively longer time periods (e.g., over hundreds of
seconds). The relatively clean background signals shown in FIGS.
20A-20B illustrate the effectiveness of the mu-metal shielding and
axial positioning. As seen in FIG. 20B, the time-averaged
background noise level as an amplitude of about 0.08 mV, which is
comparable to the noise level obtained with a piezo oscillation
actuator (see, e.g., FIG. 8D).
[0124] FIGS. 21A-21H show an example series of measurements using
the voice coil linear actuator in accordance with certain
implementations described herein. FIGS. 21A-21F show measurements
of hCG assay membranes (e.g., fabricated as described in Example 1)
with hCG concentrations from 100 ng/ml to 0.001 ng/ml in log 10
steps (see FIG. 13 for photographs of such hCG assay membranes).
FIG. 21G shows a negative control and FIG. 21H shows a measurement
with a blank membrane. These results have the same sensitivity as
do the implementation using a piezo actuator.
[0125] FIGS. 22A-22D show another example series of measurements of
Streptavidin/Biotin assay membranes (e.g., fabricated as described
in Example 3) using the voice coil linear actuator in accordance
with certain implementations described herein. FIGS. 22A-22C show
the measurements of Streptavidin/Biotin assay membranes with
printed Biotin-BSA concentrations of 10 .mu.g/ml, 1 .mu.g/ml, 0.1
.mu.g/ml, respectively, and FIG. 22D shows the measurements from a
blank sample (e.g., 0 .mu.g/ml). In particular, the clear detection
of the 0.1 .mu.g/ml sample, where the visual color of the test line
diminishes (see FIG. 17 for photographs of such strips),
demonstrates the superior sensitivity of the system 100 of certain
implementations as compared with colorimetric detection.
[0126] FIG. 23 schematically illustrates an example apparatus 10
having the magnetic field sensor 20 positioned beneath the membrane
40 in accordance with certain implementations described herein. In
certain implementations, this arrangement advantageously simplifies
the lateral flow cassette design such that the top of the membrane
40 can be exposed for simultaneous visual examination and optical
(colorimetric) detection. Although the thickness of the membrane 40
(e.g., typically between 0.1 mm-0.5 mm) reduces the signal
intensity at the magnetic field sensor 20 positioned beneath the
membrane 40, the apparatus 10 still exhibits sufficient
sensitivity.
[0127] FIGS. 24A-24D show example measurements using a magnetic
field sensor 20 positioned beneath a series of membranes 40 in
accordance with certain implementations described herein. The
membranes 40 of FIGS. 24A-24D are the same series of
Streptavidin/Biotin assay membranes that were measured in FIGS.
22A-22D with the magnetic field sensor 20 positioned above the
membrane 40. The visually invisible 0.1 .mu.g/ml test line is still
detectable, as shown in FIG. 24C, again demonstrating the high
sensitivity of certain such implementations.
[0128] Example, non-limiting experimental data are included herein
to illustrate results achievable by various implementations of the
systems and methods described herein. All ranges of data and all
values within such ranges of data that are shown in the figures or
described in the specification are expressly included in this
disclosure. The example experiments, experimental data, tables,
graphs, plots, figures, and processing and/or operating parameters
(e.g., values and/or ranges) described herein are intended to be
illustrative of operating conditions of the disclosed systems and
methods and are not intended to limit the scope of the operating
conditions for various implementations of the methods and systems
disclosed herein. Additionally, the experiments, experimental data,
calculated data, tables, graphs, plots, figures, and other data
disclosed herein demonstrate various regimes in which
implementations of the disclosed systems and methods may operate
effectively to produce one or more desired results. Such operating
regimes and desired results are not limited solely to specific
values of operating parameters, conditions, or results shown, for
example, in a table, graph, plot, or figure, but also include
suitable ranges including or spanning these specific values.
Accordingly, the values disclosed herein include the range of
values between any of the values listed or shown in the tables,
graphs, plots, figures, etc. Additionally, the values disclosed
herein include the range of values above or below any of the values
listed or shown in the tables, graphs, plots, figures, etc. as
might be demonstrated by other values listed or shown in the
tables, graphs, plots, figures, etc. Also, although the data
disclosed herein may establish one or more effective operating
ranges and/or one or more desired results for certain
implementations, it is to be understood that not every
implementation need be operable in each such operating range or
need produce each such desired result. Further, other
implementations of the disclosed systems and methods may operate in
other operating regimes and/or produce other results than shown and
described with reference to the example experiments, experimental
data, tables, graphs, plots, figures, and other data herein.
[0129] The invention has been described in several non-limiting
implementations. It is to be understood that the implementations
are not mutually exclusive, and elements described in connection
with one implementation may be combined with, rearranged, or
eliminated from, other implementations in suitable ways to
accomplish desired design objectives. No single feature or group of
features is necessary or required for each implementation.
[0130] For purposes of summarizing the present invention, certain
aspects, advantages and novel features of the present invention are
described herein. It is to be understood, however, that not
necessarily all such advantages may be achieved in accordance with
any particular implementation. Thus, the present invention may be
embodied or carried out in a manner that achieves one or more
advantages without necessarily achieving other advantages as may be
taught or suggested herein.
[0131] As used herein any reference to "one implementation" or
"some implementations" or "an implementation" means that a
particular element, feature, structure, or characteristic described
in connection with the implementation is included in at least one
implementation. The appearances of the phrase "in one
implementation" in various places in the specification are not
necessarily all referring to the same implementation. Conditional
language used herein, such as, among others, "can," "could,"
"might," "may," "e.g.," and the like, unless specifically stated
otherwise, or otherwise understood within the context as used, is
generally intended to convey that certain implementations include,
while other implementations do not include, certain features,
elements and/or steps. In addition, the articles "a" or "an" or
"the" as used in this application and the appended claims are to be
construed to mean "one or more" or "at least one" unless specified
otherwise.
[0132] As used herein, the terms "comprises," "comprising,"
"includes," "including," "has," "having" or any other variation
thereof, are open-ended terms and intended to cover a non-exclusive
inclusion. For example, a process, method, article, or apparatus
that comprises a list of elements is not necessarily limited to
only those elements but may include other elements not expressly
listed or inherent to such process, method, article, or apparatus.
Further, unless expressly stated to the contrary, "or" refers to an
inclusive or and not to an exclusive or. For example, a condition A
or B is satisfied by any one of the following: A is true (or
present) and B is false (or not present), A is false (or not
present) and B is true (or present), or both A and B are true (or
present). As used herein, a phrase referring to "at least one of" a
list of items refers to any combination of those items, including
single members. As an example, "at least one of: A, B, or C" is
intended to cover: A, B, C, A and B, A and C, B and C, and A, B,
and C. Conjunctive language such as the phrase "at least one of X,
Y and Z," unless specifically stated otherwise, is otherwise
understood with the context as used in general to convey that an
item, term, etc. may be at least one of X, Y or Z. Thus, such
conjunctive language is not generally intended to imply that
certain implementations require at least one of X, at least one of
Y, and at least one of Z to each be present.
[0133] Thus, while only certain implementations have been
specifically described herein, it will be apparent that numerous
modifications may be made thereto without departing from the spirit
and scope of the invention. Further, acronyms are used merely to
enhance the readability of the specification and claims. It should
be noted that these acronyms are not intended to lessen the
generality of the terms used and they should not be construed to
restrict the scope of the claims to the implementations described
therein.
* * * * *